Comparison of competitive exclusion with classical cleaning and disinfection on bacterial load in pig nursery units

Comparison of competitive exclusion with classical cleaning and disinfection on bacterial load in pig nursery units RESEARCH ARTICLE Open Access Comparison of competitive exclusion with classical clea[.]

R E S E A R C H A R T I C L E Open Access

Comparison of competitive exclusion with

classical cleaning and disinfection on

bacterial load in pig nursery units

K Luyckx1, S Millet2, S Van Weyenberg1, L Herman1, M Heyndrickx1,3, J Dewulf4and K De Reu1*

Abstract

Background: Colonisation of the environment of nursery units by pathogenic micro-organisms is an important factor in the persistence and spread of endemic diseases in pigs and zoonotic pathogens These pathogens are generally controlled by the use of antibiotics and disinfectants Since an increasing resistance against these

measures has been reported in recent years, methods such as competitive exclusion (CE) are promoted as

promising alternatives

Results: This study showed that the infection pressure in CE units after microbial cleaning was not reduced to the same degree as in control units Despite sufficient administration of probiotic-type spores, the analysed bacteria did not decrease in number after 3 production rounds in CE units, indicating no competitive exclusion In addition, no differences in feed conversion were found between piglets raised in CE and control units in our study Also, no differences in faecal consistency (indicator for enteric diseases) was noticed

Conclusion: These results indicate that the CE protocol is not a valuable alternative for classical C&D

Keywords: Competitive exclusion, Cleaning and disinfection, Bacterial load, Pig nursery units

Abbreviations: AC, After cleaning; AD, After disinfection; BC, Before cleaning; BPW, Buffered peptone water;

C&D, Cleaning and disinfection; CE, Competitive exclusion; CFU, Colony forming units; CS (%), Proportion of

countable samples given in percentage; D (%), Proportion of positive samples after detection given in percentage;

E coli, Escherichia coli; ILVO, Institute for agricultural and fisheries research; MBC, Minimum bactericidal

concentration; MIC, Minimum inhibitory concentration; MRSA ST398, Methicillin resistant Staphylococcus aureus sequence type 398; MRSM, chromID® MRSA-SMART medium; PIP AHC, Probiotics in process animal house cleaner; PIP AHS, Probiotics in process animal house stabilizer; Q1, First quartile; Q2, Median; Q3, Third quartile;

QAC, Quaternary ammonium compounds; TD100, Treatment days per 100 days at risk; W1, After one week of

production; W5, After five weeks of production

Background

Colonisation of the environment in nursery units by

pathogenic micro-organisms is an important factor in

the persistence and spread of endemic diseases in pigs

and of zoonotic pathogens These infections are often

controlled by the use of antibiotics and disinfectants [1]

However, an increasing level of resistance against these

substances has been observed in recent years [2–5]

Since 2005, methicillin resistantStaphylococcus aureus se-quence type 398 (MRSA ST398) has been found on farms and farm animals, especially pigs [6–8] MRSA ST398 has

a multiresistant phenotype [9], a zoonotic character [10] and can also pick up new resistance genes [11] Wong et

al [12] described the presence of disinfectant resistance genes in porcine MRSA The minimum inhibitory and bactericidal concentrations (MIC and MBC) of these MRSA strains were lower than the recommended concen-trations of disinfectants However, there is concern that an impairment of the used disinfectant, resulting in exposure

to lower active levels of these agents (e.g., due to presence

* Correspondence: koen.dereu@ilvo.vlaanderen.be

1 Institute for Agricultural and Fisheries Research (ILVO), Technology and Food

Science Unit, Melle, Belgium

Full list of author information is available at the end of the article

© 2016 The Author(s) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

of organic material), resistant MRSA strains harbouring

these disinfectant resistance genes may be selected [12]

Slifierz et al [13] showed that the use of quaternary

am-monium compound-based (QAC) disinfectants is a risk

for selecting (antibiotic resistant) MRSA in commercial

swine herds Antibiotic multiresistant Salmonella strains

on pig farms have been described in several countries

[14–16] Randall et al [17] suggested that the use of

bio-cides alone or combined with antibiotic treatment may

also increase selective pressure towards antibiotic

resist-ance ofSalmonella enterica Beier et al [18] showed that

β-haemolytic enterotoxigenic Escherichia coli (E coli)

strains isolated from neonatal pigs, were resistant to

chlor-hexidine and QAC Some of these resistant strains had

also multiple antibiotic resistance

Because of the ongoing concern about excessive use of

biocides and potential resistance development and

cross-resistance to clinically important antibiotics, the

use of bacterial biocontrol agents has often been

sug-gested as an alternative method to antagonise the

growth of these pathogens The working mechanism of

these biocontrol agents is based on the concept of

micro-organisms that should compete with pathogens in

the environment by competitive exclusion, influencing

quorum sensing, producing antimicrobial compounds

(e.g., bacteriocins) and/or competition for attachment

sites [19] However, only very few reports describing

the use and the effectiveness of microbial biocontrol

agents on farms are available in literature The aim of

this study was to compare the effectiveness of a

com-mercial competitive exclusion (CE) protocol with a

classical cleaning and disinfection (C&D) protocol in

decreasing Salmonella; (haemolytic) E coli, faecal

of nursery units during 3 successive rounds

Methods

Management in control and CE units

This study was carried out in 6 identical nursery units at

the experimental pig farm of the Institute for

Agricul-tural and Fisheries Research (ILVO) during 3 successive

production rounds Piglets were moved to these units

immediately after weaning (4 weeks of age) and stayed

there for 6 weeks Three units were assigned to the

con-trol group (classical C&D protocol) and 3 to the

treat-ment group (CE protocol) Each comparttreat-ment consists

of eight identical pens of 1.8 m2 Piglets were raised per

six in one pen After 6 weeks, piglets were transported

to fattening units and pens were cleaned (and

disin-fected) according to the tested protocols

Classical C&D protocol was carried out after pigs were

removed Manure was removed by cleaning with cold

water Twenty-four hours later, pens were soaked with

2 % MS Topfoam (sodium hydroxide) (Schippers, Bladel,

The Netherlands) for 30 min The cleaning product and any remaining dirt was removed under high pressure with cold water (150 bar) and pens were disinfected with

1 % (v/v) MS Megades (glutaraldehyde and quaternary ammonium compounds) (Schippers) Finally, the pens were kept empty during two weeks of down-time The CE units pens were first hosed down with cold water to remove manure; 24 h later they were soaked with 1.5 % (v/v) PIP AHC (Probiotics In Process Animal House Cleaner, Chrisal, Lommel, Belgium) at 40 °C for

10 min and rinsed with warm water (40 °C) PIP AHC consists of cleaning compounds, Bacillus spp spores and enzymes In CE units, no disinfection was carried out In addition, during the 2-week down-time period as well as during production, CE units were sprayed 2–3 times per week with pure PIP AHS (Animal Housing Stabilizer, Chrisal) to bring and retain biocontrol agents into the stall environment In the first week of produc-tion during the third round, CE units were sprayed every day of the week with PIP AHS The AHC and AHS PIP products containedBacillus spp spores of five different species in a concentration of 8.5 and 7.5 log colony forming units (CFU)/mL, respectively

Both protocols were carried out according to the man-ufacturers guidelines For each protocol an individual and identical high pressure jet (Kärcher, HDS 6/14-4CX, Temse, Belgium) was used

Sampling scheme

Sampling was performed at different time points (“sam-pling moments”): (1) immediately after pig loading (be-fore cleaning, BC); (2) 24 h after cleaning (CE units) (AC) or 24 h after disinfection (control units) (AD); (3) after 1 week (W1) and (4) after 5 weeks of production (W5) (piglets present) Three pens per compartment were sampled at each sampling moment Premoistened sponge swab samples with 10 mL Buffered Peptone Water (BPW) (3 M, SSL10BPW, St-Paul, USA) were taken at five locations per pen: synthetic grid floor, con-crete wall, synthetic wall, drinking nipples and feeding trough Samples were taken in triplicate per type of loca-tion resulting in 15 swab samples per nursery unit at each sampling moment After disinfection, 10 mL Dey Engley neutralising broth (Sigma Aldrich, Fluka, D3435, St-Louis, USA) was used to premoisten the sponge swab samples (SSL100, 3 M) used A surface of 625 cm2(A4 paper format) was sampled Because the surface of the drinking nipples was smaller than 625 cm2, 2 drinking nipples per pen were sampled and pooled as one sample

Sample processing

Samples were transported to the lab under refrigeration and stored at 3 ± 2 °C for 18 h before further processing Samples were first diluted with 30 ml of BPW (Oxoid,

CM0509, Basingstroke, Hampshire, England) and then

homogenized by placing them in a Masticator (IUL

in-struments, S.A., Barcelona, Spain) Prior to plating, swab

samples were further serial diluted (1:10) in peptone

physiological salt water (Bio Trading, K110B009AA,

Mijdrecht, The Netherlands) to produce countable

re-sults on the selected agar media: Slanetz-and-Bartley

(Oxoid, CM0377) for Enterococcus spp., Rapid E coli

(Biorad, 356–4024, Marnes-la-Coquettes, France) for E

coli and faecal coliforms and chromID® MRSA-SMART

(MRSM, bioMérieux, 413050,Marcy l’Etoile, France) for

MRSA enumerations Slanetz and Bartley, Rapid E coli

and MRSA-SMART agar plates were incubated at 37 °C

during 48 h, 44 °C during 24 h and at 37 °C during 24–

48 h, respectively A 3 ml BPW-fraction of the sample

was heated for 10 min at 80 °C, diluted in peptone water

and plated on Plate Count Agar (Oxoid, CM0325) for

spore enumerations in order to determine the CFU

count in both PIP products and to test if Bacillus spp

spores were well distributed and sufficiently present in

pens Plate Count Agar plates were incubated for 72 h at

30 °C Also, a 10 ml BPW-fraction of the sample was

mixed with 10 ml double concentrated Mueller Hinton

Broth (Oxoid, CM0405) and 13 % (w/v) sodium chloride

(Merck, 1.06404.500, Darmstadt, Germany) After

over-night incubation at 37 °C, 100μl was plated on MRSM for

detection of MRSA In addition, the original sample

di-luted in BPW (i.e., the remaining BPW fraction) was

over-night incubated at 37 °C for detection methods Detection

ofE coli and faecal coliforms was carried out by plating

medium Salmonella detection on the broth was carried

out according to ISO 6579:2002 Annex D protocol [20]

Confirmation of, MRSA,Salmonella and haemolytic E coli

Five positive MRSA colonies (if present) were subcultured

on Tryptone Soy Agar (Oxoid, CM0131) and DNA was

ex-tracted according to the method of Stranden et al [21] A

multiplex PCR, as described by Maes et al [22], was

per-formed for MRSA and a CC398 specific PCR, as described

by Stegger et al [23], for MRSA ST398 confirmation

Positive Salmonella colonies on Xylose Lysine

Deoxy-cholate agar medium (Oxoid, CM0469) were subcultured

on Nutrient Agar (Oxoid, CM0003) After incubation,

PCR confirmation on cel lysates was performed as

de-scribed by Aabo et al [24]

From the third down-time and production round, five

positive E coli colonies (when possible) were

subcul-tured on Columbia base Blood Agar (Oxoid, CM0331)

with 5 % sheep blood and incubated for 24 h at 37 °C

for analysis of haemolytic E coli If a plate was negative

after 24 h, it was incubated for a further 24 h To

calcu-late the enumerations of haemolyticE coli, the ratio of

the number of positive haemolyticE coli colonies on the

5 selected colonies was multiplied by the mean E coli enumeration of that sample

Other analyses

Piglets were weighed individually at the age of 4, 6 and

9 weeks Also feed intake was monitored per pen on the same moments allowing to calculate feed conversion ratio

of every pen

In addition, faecal consistency was evaluated according

to [25]: a score from 1 (no diarrhea) to 4 (serious diarrhea) was assigned per pen

Finally, clinical manifestations and treatment with an-tibiotics were registered Treatments days per 100 days

at risk (TD100) was calculated per pen for each proto-col This was done by calculating the ratio of treatments days (number of days that piglets received antibiotics) and the number of days at risk (time that pigs could be exposed to antibiotics), taking the number of dead pig-lets into account This ratio was then multiplied by 100

Statistical analysis

The distribution of the dependent variables was charac-terised with a histogram and Q-Q plot Log transformed enumerations of spores and Enterococcus spp and results

of average daily gain, daily feed intake, feed conversion ratio and TD100 ratio followed a normal distribution Log transformed enumerations of E coli, haemolytic E coli, faecal coliforms and MRSA did not follow this distribution The 4 point scale faecal consistency score was reduced

to a binary scale: 0 = pens with score 1 and 1 = pens with score > 1

The effect of the predictor variables on the normal distributed data (dependent variables) was assessed using multivariate linear regression The effect of predictor variables on the non normally distributed outcome vari-ables describing the enumeration and detection of the different bacteria (absence or below the detection limit =0, presence =1) was tested by means of multi-variate logistic regression analysis

A backward stepwise elimination was performed to determine the final statistical model for each bacterio-logical parameter, starting with the global model (predictor variables: protocol used, sampling moment, production round and location) and subsequently re-moving all non-significant terms Only biologically rele-vant interaction effects were considered In each model, the variables compartment and pen were included as a random effect to correct for measurements within one pen and compartment The predictor variable sampling moment was included as a repeated measure Post-hoc comparison was performed with a Tukey-Kramer test Throughout the analyses, P-values ≤ 0.05 were consid-ered as significant

All statistical analyses were carried out with Statistical

Analysis System software (SAS®, version 9.4, SAS

Insti-tute Inc.)

Results

In total 1074 swab samples were taken during 3

succes-sive rounds At each sampling moment approximately

90 samples were taken: i.e., 45 in CE units (n = 3) and 45

in control units (n = 3)

Spore enumerations

At every sampling moment and in each production round,

higher spore enumerations were found for CE units

com-pared to control units (P < 0.01) (Fig 1a and b), with a

minimal difference of 0.70 log (BC) and 1.15 log (first

round) CFU (colony forming units)/sampling area

Fur-ther, spore enumerations increased after every round in

CE units (P < 0.01) (Fig 1b) Mean spore enumerations

ranged from 2.88 log CFU/sampling area AC to 4.89 log

CFU/sampling area at W5 during production piglets

present and from 1.25 log CFU/sampling area AD to 2.61

log CFU/sampling area at W5 for CE and control units,

respectively

Enterococcus spp enumerations

When considering the overall contamination level in

both units, higher Enterococcus spp enumerations, with

a mean difference of 0.80 log CFU/sampling area, were

found in CE units (P < 0.01) After disinfection of control

units, lower Enterococcus spp enumerations were

ob-served compared to cleaned CE units (P < 0.01) (Fig 2a)

The mean difference was 2.88 log CFU/sampling area Cleaning of CE units caused a reduction of 0.42 log CFU/sampling area, while in disinfected control units a reduction of 3.54 log CFU/sampling area was noticed Before cleaning and after 1 week of production, no dif-ferences in Enterococcus spp enumerations were found between units However, at W5, higherEnterococcus spp enumerations were found in CE units (P = 0.05) In addition,Enterococcus spp enumerations were higher in every production round for CE units (P < 0.01) (Fig 2b)

E coli enumerations

More E coli countable samples were found for CE units after cleaning compared to control units after disinfec-tion (P < 0.01) (Fig 3a) Propordisinfec-tion of countable samples was reduced by 9 % AC of CE units, while a reduction

of 41 % was obtained after disinfecting control units During production and before cleaning, no differences were found in amount of countable E coli samples be-tween both types of units

In control units, lower amounts of countable samples were found AD compared to amounts found BC and W1 (P < 0.01) while this was not seen AC of CE units (Fig 3a) Descriptive values of E coli enumeration at each sam-pling moment are given in Table 1

HaemolyticE coli enumerations

Of all samples taken in CE units (n = 180) and control units (n = 180) during the 3rd round, 24 % and 23 % were positive for haemolytic E coli, respectively Of these positive samples, 16 % were obtained AC (CE

Fig 1 Mean spore enumerations in log colony forming units/sampling area for CE (dark grey bars) and control units (light grey bars) At each sampling moment (a) and per round (b), 135 and 180 samples were taken per unit type, respectively Significant differences between sampling moments or rounds within one type of unit are indicated by different letters above bars Significant differences between protocols within one sampling moment or round are indicated by a star (*) on the horizontal axis Vertical bars denote standard errors BC, before cleaning; AC/AD, after cleaning (CE unit) or after disinfection (control unit); W1, after 1 week of production; W5: after 5 weeks of production

units) and 0 % were obtained AD (control units),

respect-ively Mean enumerations were 3.0 log CFU/sampling area

for both types of units No significant differences were

noticed between units

Faecal coliform enumerations

When comparing CE and control units, results of faecal

coliform enumeration confirmed the observations

ob-tained with E coli analyses (Fig 3c) A reduction of 26

and 51 % of faecal coliform countable samples was

ob-tained AC and AD of CE and control units, respectively

After cleaning as well as AD, a significant reduction of

faecal coliform countable samples was obtained (P < 0.01)

Faecal coliform enumerations at each sampling

mo-ment for both types of units are given in Table 1

E coli and faecal coliform detection

Detection results ofE coli (Fig 3b) and faecal coliforms

(Fig 3d) confirmed the enumeration results of both

parameters

MRSA enumerations

After cleaning, countable samples were reduced 61 % for

CE units, 20 % less than the observed reduction in

disin-fected control units (P < 0.01) (Fig 3e) When pens were

soiled (BC, W1 and W5), no differences in MRSA

con-tamination were found between both types of units

Mean and median enumerations for MRSA are given

for each sampling moment in Table 1

MRSA detection

Detection results showed that the number of MRSA positive samples was the highest (90 %) for CE units compared to the control units (81 %) (P < 0.01) (Fig 3f)

Salmonella detection

NoSalmonella was found in this study

Sampling locations

Mean enumerations (with standard deviation) and me-dian enumerations (with first and third quartile) of En-terococcus spp., E coli, faecal coliforms and MRSA after cleaning (CE units) and disinfection (control units) are given per type of sampling location in Table 2 In addition, the percentage of countable swab samples (enumerations) and positive samples after enrichment (detection) is shown for both types of units Also, mean spore andEnterococcus spp counts on all samples taken

in CE and control units are given for each type of loca-tion in Figs 4 and 5, respectively

After cleaning of CE units, enumerations of Entero-coccus spp were the highest for floors, concrete walls and drinking nipples In addition, highest percentage of

were found for floors and concrete walls Moreover, after enrichment also drinking nipples were still often con-taminated with E coli Results of faecal coliforms and MRSA confirmed these observations

In control units, high numbers of Enterococcus spp

Fig 2 Mean Enterococcus spp enumerations in log colony forming units/sampling area for CE (dark grey bars) and control units (light grey bars).

At each sampling moment (a) and per round (b), 135 and 180 samples were taken per unit type, respectively Significant differences between sampling moments or rounds within one type of unit are indicated by different letters above bars Significant differences between protocols within one sampling moment or round are indicated by a star (*) on the horizontal axis Vertical bars denote standard errors BC; before cleaning, AC/AD, after cleaning (CE unit) or after disinfection (control unit); W1, after 1 week of production and W5: after 5 weeks of production

coli positive samples after enrichment were found for

floors, drinking nipples and feeding troughs In

addition, highest enumerations for faecal coliforms

were also found at these locations Finally, for MRSA, drinking nipples were the most contaminated after disinfection

Fig 3 Percentage of positive samples before (enumerations) and after enrichment (detection) for E coli (a-b), faecal coliforms (c-d) and

MRSA (e-f) given for CE (dark grey bars) and control units (light grey bars) At each sampling moment and in total 135 and 540 samples were taken per unit type, respectively Significant differences between sampling moments within one type of unit are indicated by letters above bars Significant differences between protocols within one sampling moment are indicated by a star (*) on the horizontal axis BC, before cleaning; AC/AD, after cleaning or after disinfection; W1, after 1 week of production and W5: after 5 weeks of production

More spore enumerations were found at every location for CE units (Fig 4), with a minimal difference of 1.2 log CFU/sampling area

In addition, when considering the overallEnterococcus spp contamination level, higher levels were found for each location in CE units (Fig 5)

Performance results

Mean starting weight of piglets in CE and control pens was 7.4 ± 1.5 and 7.1 ± 1.5 kg, respectively A mean feed intake of 0.539 ± 0.078 and 0.521 ± 0.065 kg/day was ob-served for CE and control units, respectively No signifi-cant differences were found between feed intake of piglets raised in CE and control pens When considering results of daily gain, no significant differences were found Average daily gain was 0.407 ± 0.056 and 0.395 ± 0.053 kg for piglets in CE and control pens, respectively

In addition, no significant differences in mean feed con-version were found: 1.327 ± 0.072 and 1.324 ± 0.085 for pigs in CE and control units, respectively

Faecal consistency

No significant differences in scores of faecal consistency between protocols were noticed (data not shown)

Table 2 Descriptive values for Escherichia coli (E coli), faecal coliforms and methicillin resistant Staphylococcus aureus (MRSA) enumerations (log colony forming units/sampling area) and detection after cleaning (CE units) and disinfection (control units) for each type of sampling location Detection method was carried out after an overnight enrichment of samples

CE units

Control units

Mean and standard deviation are given for enumerations that are normally distributed First quartile (Q1), median (Q2, bold characters) and third quartile (Q3) are given for enumerations that did not follow this distribution

a

1, floors

b

2, concrete walls

c

3, synthetic walls

d

4, drinking nipples

e

5, feeding trough

f

CS (%), proportion of countable samples given in percentage

g

Table 1 Descriptive values for Escherichia coli (E coli), faecal

coliforms and methicillin resistant Staphylococcus aureus (MRSA)

enumerations (log colony forming units/sampling area) given for

each sampling moment for CE units and control units

CE units

Control units

Mean and standard deviation are given for enumerations that are normally

distributed First quartile (Q1), median (Q2, bold characters) and third quartile

(Q3) are given for enumerations that did not follow this distribution

a

BC, before cleaning

b

AC/AD, after cleaning/after disinfection

c

W1, after 1 week of production

d

W5, after 5 weeks of production

Antibiotic treatment

The mean TD100 for CE and control units was 27.9 ±

0.9 and 28.3 ± 2.1 %, respectively No significant

differ-ences were found between protocols

Discussion

The emergence of multiresistant (pathogenic) bacteria is

of great concern for animal and human health Excessive

use of antibiotics [26, 27] and disinfectants [28–30] in

for example the animal primary production, could

pos-sibly contribute to this phenomenon Therefore,

alterna-tive methods such as competialterna-tive exclusion (CE) are

promoted as promising In this study a commercial CE

protocol (by probiotic-type bacteria) was compared with

a classical C&D protocol in nursery units

According to the manufacturer of the PIP products, a reduction of pathogenic bacteria and improvement in hygiene after CE during 3 successive production rounds should be obtained The first statement could not be confirmed by this study: E coli (Salmonella-indicator), haemolytic E coli and MRSA analyses showed that the infection pressure after CE cleaning was not reduced to the same extent as implementing a disinfection step Furthermore, during production no differences were no-ticed Also no improvement in hygiene was seen: during

enumerations (hygiene indicator) and no differences in

Fig 5 Mean Enterococcus spp enumerations in log colony forming units/sampling area for CE (dark grey bars) and control units (light grey bars) for each location At each location, 108 samples were taken per type of unit Significant differences between sampling moments within one type of unit are indicated by different letters above bars Significant differences between protocols within one sampling moment are indicated by a star (*) on the horizontal axis Vertical bars denote standard errors 1, grid floor; 2, concrete wall; 3, synthetic wall; 4, drinking nipples; 5, feeding trough

Fig 4 Mean spore enumerations in log colony forming units/sampling area for CE (dark grey bars) and control units (light grey bars) for each location At each location, 108 samples were taken per type of unit Significant differences between sampling moments within one type of unit are indicated by different letters above bars Significant differences between protocols within one sampling moment are indicated by a star (*) on the horizontal axis Vertical bars denote standard errors 1, grid floor; 2, concrete wall; 3, synthetic wall; 4, drinking nipples; 5, feeding trough

faecal coliforms (faecal indicator) contamination

be-tween the two types of units were found Because, higher

contamination levels of MRSA and pathogen-indicator

organisms (E coli) were found in CE units after cleaning,

there may be a greater chance of infecting young piglets

arriving in those nurseries

Several hypotheses have been proposed to explain the

mechanisms of CE cultures One is that CE bacteria

should compete with other bacteria for adhesion sites,

nutrients and energy, which results in preventing growth

and proliferation of pathogenic bacteria in the

environ-ment (Cummings and Macfarlane, [31]) Another

hy-pothesis is that these bacteria influence the quorum

sensing communication and therefore inhibit expression

of virulence and colonisation genes of pathogens (Vilà et

al [32]; Deep et al [33] ) Besides CE bacteria, also

en-zymes were administered during cleaning, with the aim

of helping to eliminate biofilms In this study, no

reduc-tion of the analysed bacteria after 3 producreduc-tion rounds

in CE units was seen Several explanations were found

to clarify this observation: (i) adhesion sites are

abun-dantly present in animal houses, hence there is no need

for competition; (ii) removal of organic debris is only

carried out when piglets are removed from pens,

there-fore CE-, pathogenic and other bacteria have an

abun-dance of nutrients during production, eliminating the

need for competition between bacteria; (iii) however, in

order to compete for nutrients, spores need to

germin-ate, which may not be the case for all spores

Moreover, Luyckx, et al [34] (i.e., chapter III) showed

that a cleaning step in broiler houses caused a reduction

of total aerobic bacteria with 2 log CFU/sampling

sur-face and that a disinfection step caused a further

reduc-tion of 1.5 log CFU Although, cleaning caused a greater

reduction of total aerobic bacteria, both the above study

and this one showed that a disinfection step is still an

important step for further reducing the bacterial

infec-tion pressure in barns with naturally high levels of

envir-onmental bacteria

Improvement of feed conversion efficiency by

probiotic-type bacteria could be obtained by a shift in intestinal

flora, stimulating growth of nonpathogenic facultative

anaerobic bacteria, inhibiting growth of pathogens, and

enhancing digestion and utilisation of nutrients [35]

However, no differences were found between piglets raised

in CE and control units in our study Also, no differences

in faecal consistency was noticed A possible explanation

could be that not enough CE bacteria could be

adminis-tered directly to the animals through the environmental

spray application

Finally, the contamination levels of the different

sam-pling locations were analysed after cleaning of CE units

and disinfection of control units In CE units, grid floors,

concrete walls and drinking nipples were still mostly

contaminated by Enterococcus spp., E coli, faecal coli-forms and MRSA after cleaning Although spore counts showed that high numbers of CE bacteria were present

at these locations, the contamination level of different bacteria was still much higher compared to the micro-bial load after disinfection of control units In addition, the overall Enterococcus spp contamination of all loca-tions during the experiment was higher in CE units In control units, grid floors and drinking nipples seemed critical locations after disinfection Luyckx, et al [36] also showed that drinking cups are critical locations for C&D in broiler houses

A limitation of our study was that the CE protocol was only carried out in pig nursery units, and not in farrow-ing units Therefore, the piglets gut microbiota was already formed, which could contain pathogens and con-taminate pig nursery units on arrival Conversely, this is also a drawback of the CE protocol A future perspective could be to determine the efficacy of a CE protocol ap-plied on the whole farm, however this approach would substantially increase the work load and associated costs for the farmer

Conclusions

Very few studies about the impact of microbial cleaning and administration during production on the environ-ment in animal houses are available Our results showed that competitive exclusion by probiotic-type bacteria could not meet the claims provided by the manufacturer Moreover, this study showed that a good C&D protocol during down-time is still very important for reducing in-fection pressure in nursery units However, more re-search should be carried out for a valuable alternative, because disinfectant resistance might be an upcoming problem

Acknowledgments Many thanks go to Kristof Dierkens and Eline Dumoleijn for their practical support We also thank Miriam Levenson for the English language editing of this manuscript.

Funding This research is funded by the Belgian Federal Public Service for Health, Food Chain Safety and Environment (RT 11/8 Cleandesopt).

Availability of data and materials The datasets supporting the conclusions of this article are included within the article.

Authors ’ contributions

KL was involved in the sample collection, laboratory analyses, statistical analyses, interpretation of the data and drafting the manuscript JD, SM and

KD coordinated the study SVW and JD evaluated the statistical analyses KL,

SM, SVW, LH, MH, JD and KD contributed to development and writing of the paper All authors read and approved the final manuscript.

Competing interests The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Author details

1 Institute for Agricultural and Fisheries Research (ILVO), Technology and Food

Science Unit, Melle, Belgium 2 Institute for Agricultural and Fisheries Research

(ILVO), Animal Sciences Unit, Melle, Belgium 3 Department of Pathology,

Bacteriology and Poultry Diseases, Ghent University, Faculty of Veterinary

Medicine, Merelbeke, Belgium 4 Veterinary Epidemiology Unit, Department of

Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine,

Ghent University, Merelbeke, Belgium.

Received: 27 January 2016 Accepted: 26 August 2016

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